Field of the Invention
Protein-based binding reagents have various uses in therapeutic or diagnostic application. Antibodies have proven to be an excellent paradigm for such reagents. Indeed, a number of monoclonal antibodies (mAbs) have been successfully used for treating cancers, infectious diseases, and inflammatory diseases (Adams et al., Nat. Biotechnol. 2005 September; 23:1147-57.).
Description of the Related Art
Antibody affinity is a key factor in the success of an antibody as a therapeutic agent. An antibody with high affinity allows the antibody to compete effectively with the natural ligand for the targeted receptor to reduce dosage, toxicity, and cost. Multimerization of antigen binding sites has been shown to be an effective means of increasing the overall strength of the binding of an antibody to an antigen which is defined as the antibody avidity (functional affinity) (Miller et al., J Immunol 170:4854-4861, 2003; Rheinnecker et al., J Immunol 157:2989-2997, 1996; Shopes, J Immunol 148:2918-2922, 1992; Shuford et al., Science 252:724-727, 1991; Wolff et al., J Immunol 148:2469-2474, 1992). Multivalent antibodies have increased antitumor activity in vivo (Liu et al., Int Immunopharmacol 6:79 1-799, 2006; Wolff et al., Cancer Res 53:2560-2565, 1993). Due to the bivalent nature of immunoglobulin G (IgG), conventional and engineered IgG cannot be used for simultaneous binding to more than two different antigens. Thus, there is a need for multi-valent or multi-specific protein-based binding reagents.
In some cases, avoiding the effector function, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC), through engineering the Fc region is necessary to reduce mitogenicity side-effects. For example, the murine anti-human CD3 mAb (Orthoclone OKT3, muromonab-CD3), is a potent immunosuppressive agent targeting the T-cell receptor (TCR/CD3 complex on human T cells. It has been used during the last two decades to prevent or treat allograft rejection (Cosimi et al., N Engl J Med 305:308-314, 1981; Group, N Engl J Med 313:337-342, 1985; Kung et al., Science 206:347-349, 1979). However, one major drawback to the use of this therapy is the systemic release of cytokines such as TNF-α, IL-2, and IFN-γ, which result in a series of adverse mitogenic effects, including flu-like symptoms, respiratory distress, neurological symptoms, and acute tubular necrosis (Abramowicz et al., Transplantation 47:606-608, 1989; Chatenoud et al., N Engl J Med 320:1420-1421, 1989; Goldman et al., Transplantation 50:158-159, 1990; Toussaint et al., Transplantation 48:524-526, 1989). Since the mitogenic activity of OKT3 and other anti-CD3 mAbs depends upon extensive TCR/CD3 cross-linking via binding to FcR-positive cells (e.g. monocytes), recent efforts have been devoted to developing nonmitogenic forms of anti-CD3 antibodies by altering binding to FcR. Thus, there is a need for protein-based binding reagents that have high affinity, low mitogenic effect, and high in vivo stability.
Collagen is the most abundant protein in mammals. It is an extracellular matrix protein that contains one or more triple-helical regions (collagenous domains) with a repeating triplet sequence Gly-X-Y, where X and Y are frequently proline (amino acid code, P or Pro) and hydroxyproline (amino acid code, O or Hyp). The presence of such triplets allows three collagen polypeptide chains (α-chains) to fold into a triple-helical conformation. Many collagen-like proteins with collagenous domains are present in human serum and serve as an innate immune system in protection from infectious organisms. These include complement protein C1q, macrophage receptors, collectin family proteins-mannose binding lectin (MBL), ficolins and surfactant proteins A and D (SP-A and SP-D). A common structural feature among these “defense collagen” molecules is that all of them are in multi-trimeric protein units with a target-binding domain at the C-terminus. Consequently, multimerization significantly increases the functional affinity of the binding domain of these defense collagen molecules.
Trimerization of heterologous fusion proteins containing collagenous domain(s) has been accomplished by employing either a homogeneous or heterologous trimerization domain fused to the collagenous domain to drive the collagen triplex formation. Examples of a trimer-oligomerizing domain include a C-propeptide of procollagens, a coiled-coil neck domain of collectin family proteins, a C-terminal portion of FasL and a bacteriophage T4 fibritin foldon domain (Frank et al., (2001) J Mol Biol 308: 1081-1089; Holler et al., (2003) Mol Cell Biol 23: 1428-1440; Hoppe et al., (1994) FEBS Lett 344: 191-195).
The trimeric assembly of fibrillar collagens (types I, II, III, IV, V, and XI) and collectin family proteins are initiated by trimeric association of their large globular C-terminal domains (C-propeptides, ˜250 amino acids) and C-terminal coiled-coil neck domains (˜35 amino acids), respectively, following by propagation of the collagen domain(s) in a zipper-like fashion from the C to the N terminus (Bachinger et al., (1980) Eur J Biochem 106: 619-632; Hakansson et al., (1999) Structure 7: 255-264; Hakansson and Reid, (2000) Protein Sci 9: 1607-1617; Prockop and Kivirikko, (1995) Annu Rev Biochem 64: 403-434; Sheriff et al., (1994) Nat Struct Biol 1: 789-794; Weis and Drickamer, (1994) Structure 2: 1227-1240).
The sequence Gly-Pro-Hyp is the most stabilizing and most common triplet in collagen and the peptide (Gly-Pro-Hyp)10 (SEQ ID NO: 19) can self-associate into a highly stable triple helical structure (Chopra and Ananthanarayanan, (1982) Proc Natl Acad Sci USA 79: 7180-7184; Engel et al., (1977) Biopolymers 16: 601-622; Sakakibara et al., (1973) Biochim Biophys Acta 303: 198-202; Yang et al., (1997) J Biol Chem 272: 28837-28840). In contrast to chemically synthesized (Gly-Pro-Hyp)10 (SEQ ID NO: 19) peptide, the (Gly-Pro-Pro)10 (SEQ ID NO: 20) peptide does not self-assemble into a stable triple-helix under physiological conditions (Engel et al., (1977) Biopolymers 16: 601-622). For obtaining a thermally stable (Gly-Pro-Pro)10 (SEQ ID NO: 20) triplex, two approaches have been described. First, a interchain disulfide-bonded (Gly-Pro-Pro)10 (SEQ ID NO: 20) triplex was obtained in vitro by a redox-shuffling process of a disulfide knot of type III collagen either C- or N-terminal adjacent to the collagen-like peptide at 20° C. (Boudko et al., (2002) J Mol Biol 317: 459-470; Frank et al., (2003) J Biol Chem 278: 7747-7750). Second, a stable heterologous trimerizing foldon domain derived from bacteriophage T4 fibritin was fused to the C-terminus of (Gly-Pro-Pro)10 (SEQ ID NO: 20) peptide to drive the trimerization and correct folding of the collagen-like peptide in a P4H-deficient E. coli expression system (Frank et al., (2001) J Mol Biol 308: 1081-1089). Many studies have examined the melting temperatures/stability of G-X-Y repeats. Frank et al., (2001); Persikov et al., (2000) Biochemistry 39, 14960-14967; Persikov et al., (2004) Protein Sci. 13: 893-902; and Mohs et al., (2007) J. Biol. Chem. 282: 29757-29765. Based on these studies, the stability of various repeat structures can be predicted.
The approaches described above are limited in their use because they may not support normal trimerizing and folding of a heterologous polypeptide, and may introduce a hetero-antigenetic fragment associated with the risk of an immune response that could severely limit potential therapeutic applications. Thus, what is needed is an in vivo expression system capable of forming a thermally stable triple helical structure that drives the formation of a trimeric fusion protein, enabling use of such trimerized polypeptides both in vitro and in vivo.
The recombinant expression of collagens and hydroxyproline-containing peptides with functional triple-helix conformation requires specific post-translational enzymes, in particular prolyl 4-hydroxylase (P4H) (Prockop and Kivirikko, (1995) Annu Rev Biochem 64: 403-434). Prolines specified in the Y position of Gly-X-Y motif of collagen are generally post-translationally modified to 4-hydroxyproline by prolyl 4-hydroxylase (P4H) to stabilize the triple-helical structure of collagen. In the absence of proline hydroxylation, the essential triple helical conformation of collagen is thermally unstable at below physiological temperatures (Berg and Prockop, (1973) Biochem Biophys Res Commun 52: 115-120; Rosenbloom et al., (1973) Arch Biochem Biophys 158: 478-484). Procaryotes do not possess any P4H activity. Yeasts and insect cells exhibit insufficient enzyme activity to achieve recombinant collagen expression unless exogenous P4H genes (both α and β subunits) are introduced simultaneously to form an active α2β2 tetramer.
The non-fibrillar FACIT (fibril-associated collagen with interrupted triple-helices) collagens (types IX, XII, XIV, XVI, XIX, XX, XXI and XXII) are a subgroup within the collagen family. They appear to connect with fibrillar collagens and other matrix components or cells (Shaw and Olsen, (1991) Trends Biochem Sci 16: 191-194). In FACITs, the two conserved cysteines, separated by four amino acids, are located at the junction of the COL1 and NC1 domains and are responsible for interchain disulfide bonding among the three assembled collagen chains (Mazzorana et al., (2001) J Biol Chem 276: 27989-27998), which is hereby specifically incorporated by reference in its entirety.
Type XII and XXI minicollagens comprising the extreme C-terminal collagenous (COL1) and noncollagenous (NC1) domains, along with the two subunits of human P4H genes have been co-expressed in a baculovirus-infected Trichoplusia ni and Drosophila S2 insect cells, respectively (Mazzorana et al., (2001) J Biol Chem 276: 27989-27998; Li et al., (2005) Biochem Biophys Res Commun 336: 375-385). Formation of interchain disulfide-bonded minicollagen XII and XXI depends on the hydroxyproline content of collagen chains, suggesting that the folding of the triple helix precedes the formation of the disulfide bonds. Insufficient prolyl hydroxylation in minicollagen XXI leads to the production of interchain disulfide-bonded dimers and intrachain disulfide-bonded monomers (Li et al., (2005) Biochem Biophys Res Commun 336: 375-385). Constructs containing the entire COL1 domain of chicken collagen XII could form trimers. Mazzorana et al. have shown that constructs containing the entire NC1 domain of chicken collagen XII and the only the five terminal G-X-Y repeats of the COL1 domain could not form trimers. The presence of five additional C-terminal G-X-Y repeats of the COL1 domain allowed the formation of trimers. The constructs used by Mazzorana contained a short fragment of human c-myc protein as a tag. As such, Mazzorana did not address the effect of trimerization with these sequences on the folding or functionality of an attached molecule or the effect of a larger attached molecule on self-trimerization.